The biosensor field is on a continuous quest for ever-greater sensitivity. In conventional bioassays, where the signal is directly proportional to the target concentration, the sensitivity is determined by the intrinsic target affinity of the bioreceptor being used for detection. In this scenario, it would be difficult to generate a measurable signal at target concentrations more than 100-fold lower than the dissociation constant (KD) of the bioreceptor.
Small molecules are important targets with the potential of clinical or commercial applications. Thus, efforts to develop methods for portable, low-cost and quantitative detection of a broad range of small molecules on site are gaining momentum. Methods that are highly sensitive and selective, including high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), fluorescence and electrochemistry, have been used for the detection of small molecules. However, these methods are time-consuming and require expensive reagents, advanced equipment, complex sample preparation, and trained operators. As a result, colorimetric methods have attracted interest for on-site and real-time detection of small molecules, due to their unique properties including cost-efficiency, lack of instrumentation, rapidness, and simplicity.
Aptamers are nucleic acid-based molecules that can be isolated in vitro through systematic evolution of ligands by exponential enrichment (SELEX) processes to bind various targets with high specificity and affinity,1 including proteins, metal ions, small molecules, and even whole cells. They are increasingly being used as recognition elements in biosensing platforms due to their low cost of production, ease of modification, chemical stability, and long shelf life.2,3 The generation of a sensor readout typically requires an aptamer to undergo some manner of binding-induced change, and most aptamer-based sensors utilize structure-switching aptamers that undergo a major conformational rearrangement upon target binding. However, most aptamers do not innately undergo a measureable binding-induced conformational change, and the development of a structure-switching aptamer typically entails a multi-stage process of sequence analysis and chemical modification.
After identifying the target-binding domain of a newly-isolated aptamer through truncation,4 structure-switching functionality is then introduced via methods such as sequence engineering,4 splitting,5 or utilization of a complementary strand.6 These methods are laborious and require considerable trial and error. Additionally, engineered structure-switching aptamers often have low target-binding affinities, which limit their utility.
SELEX methods have been developed that make it possible to isolate aptamers with inherent structure-switching functionality. For example, Ellington7 and Li8 utilized a ‘strand-displacement’ strategy to directly isolate structure-switching aptamers. Their approach begins with the hybridization of library molecules with an immobilized complementary strand. Library strands that bind to the target undergo a conformational change, dissociate from the complementary strand, and are collected in the supernatant. After several rounds of isolation and enrichment, these aptamers are sequenced, chemically modified with a fluorophore, and can then be directly employed in strand-displacement assays with a quencher-modified complementary strand.
This approach eliminates the need for sequence engineering, but there is an inherent conflict between the requirements for aptamer isolation and sensor development. This is because the isolation of high-affinity aptamers requires strong hybridization of the complementary strand with the library molecules in order to create an energetic barrier for stringent selection. However, since the complementary strands have high binding affinity for the aptamer, they inadvertently inhibit target-binding and consequently reduce the sensitivity of strand-displacement assays. This is particularly problematic for the detection of small-molecule targets, which usually have low affinity for aptamers. As such, the limit of detection of strand-displacement assays for small molecules is usually comparable to or even higher than the dissociation constant of the employed aptamer. Moreover, such assays require a heating-and-cooling process lasting ≥30 min to ensure complete hybridization between the complementary strand and aptamer to achieve low background. This time-consuming step greatly hinders the use of these assays for rapid on-site detection. Nonetheless, the strand-displacement assay is presently the only generally applicable method for developing aptamer-based small-molecule sensors directly from isolated aptamers without further engineering.
Synthetic cathinones (also known as ‘bath salts’) are designer drugs sharing a similar core structure with amphetamines and 3,4-methylenedioxy-methamphetamine (MDMA). They are highly addictive central nervous system stimulants, and are associated with many negative health consequences, including even death. Although these drugs have emerged only recently, abuse of bath salts has become a threat to public health and safety due to their severe toxicity, increasingly broad availability, and difficulty of regulation. More importantly, there is currently no reliable presumptive test for any synthetic cathinone. Chemical spot tests used to detect conventional drugs such as cocaine, methamphetamine, and opioids show no cross-reactivity to synthetic cathinones.
Therefore, there is a need for methods and materials to rapidly and selectively detect small molecules such as synthetic cathinones, in particular, for clinical or field setting.